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Abstract

The present invention relates to two ATP diphosphohydrolases (ATPDase enzymes) isolated from bovine aorta and pig pancreas, which enzymes have a molecular weight for their catalytic unit of about 78 and 54 Kilodaltons, respectively. A first process for obtaining a highly purified ATPDase is also an object of the present invention. This process has been successfully applied to the purification of both the pancreatic and the aorta enzymes and is deemed to work in the purification of any ATPDase. For both sources of enzymes, the process allows the specific activity of the enzyme to be increased by at least 10,000 fold when compared to the activity retrieved in the crude cell homogenates. The novel process involves an ion exchange chromatography step, a separation on an affinity column, followed by an electrophoresis under non-denaturing conditions. The two enzymes purified by this process (aortic and pancreatic) are glycosylated and, when deglycosylated, have molecular weights shifted to about 56 and 35 Kdaltons, respectively. Partial amino acid sequences have been obtained for each enzyme. The partial sequences appear highly homologous with a human lymphoid cell activation antigen named CD39. An antibody directed against the porcine pancreatic enzyme cross-reacts with a protein present in endothelial cell lines and in bovine aorta (78 KDa). The high degree of homology of the pancreatic and aortic enzymes with CD39 and their cross-reactivity are indications that both enzymes are related. The pancreatic enzyme completely lacks the first 200 amino acids of CD39, which means the ATPDase activity is comprised between residues 200 and 510 of CD39. Since this is the first time that a sequence is assigned to ATPDases, a second new process for producing ATPDases by recombinant technology can also be used. Therefore a second new process for producing an ATPDase using the CD39-encoding nucleic acid or part or variant thereof is also described.

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First Claim